Figure 5. Role of Ser³⁵⁸ phosphorylation in adipocytes.

(A) 3T3-L1 adipocytes were stimulated with forskolin (50 μM, 15 min; Fsk) or insulin (100 nM, 10 min; Ins) and phosphorylation of endogenous SIK2 was analysed using a phosphospecific antibody targeting Ser³⁵⁸. Quantified phosphorylation data are presented as means±S.D. for two individual experiments. (B) Adenoviral vectors (Ad) encoding Ser358Ala or Ser587Ala mutant HA–SIK2 were constructed and used to evaluate the role of Ser³⁵⁸ phosphorylation in primary rat adipocytes. Wild-type (Wt), Ser358Ala (S358A) and Ser587Ala (S587A) mutant HA–SIK2 expressed in CL 316,243-treated (100 nM, 30 min) rat adipocytes were analysed with regard to phosphorylation (upper panel, pPKA substrate and phosphoSer³⁵⁸) and 14-3-3 binding (lower panel, co-immunoprecipitation and far-Western blot). Quantified data are presented as means±S.D. for two individual experiments. Blots shown are representative of two to four experiments.